The present invention relates to 5,6-dihydro-xcex1-pyrones useful as cytokine production inhibitors, to the preparation of these compounds and to pharmaceutical and veterinary compositions containing them.
We have now discovered that fermentation of a strain of the fungus Phomopsis sp. in a nutrient medium produces two 5,6-dihydro-xcex1-pyrones esterified in the 5-position with an unsaturated C14 fatty acid and also the free C14 fatty acid. We have also discovered that fermentation of a strain of the fungus Paecilomyces sp. in a nutrient medium produces a useful phomalactone.
The present invention therefore provides a 5,6-dihydro-xcex1-pyrone of formula (I): 
wherein R is CO2H or CH3 and each of R1 and R2 is H; or R is CO2H, one of R1 and R2 is H and the other is OH; or, when R is CO2H, a pharmaceutically or veterinarily acceptable salt thereof.
Preferred compounds of the invention are:
3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-enoic acid; and
3-((5S,6S)-5,6-dihydro-5-((6S)-4,6-dimethyldodeca-2E,4E-dienoyl)-2H-pyran-2-on-6-yl)-prop-2E-ene.
The present invention provides a process for the preparation of a 5,6-dihydro-xcex1-pyrone of formula (I) or a pharmaceutically or veterinarily acceptable salt thereof, which process comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof which produces a said 5,6-dihydro-xcex1-pyrone;
(ii) isolating a said 5,6-dihydro-xcex1-pyrone from the fermentation broth; and
(iii) if desired, when the isolated 5,6-dihydro-xcex1-pyrone is the compound of formula (I) wherein R is CO2H, converting the said 5,6-dihydro-xcex1-pyrone into a pharmaceutically or veterinarily acceptable salt thereof.
The compounds of formula (I) have been isolated from a microorganism which we have designated X22502 and which has been identified as a strain of the genus Phomopsis (Saccardo) Bubxc3xa1k on the basis of the following morphological data with reference to the description given by SUTTON, B. C., 1980 (The Coelomycetes. Farnham Royal: Commonwealth Agricultural Bureaux):
The fungal strain Phomopsis sp. (X22502) (CBS 313.96) is a coelomycete isolated from tropical freshwater foam which produces a dense, dark grey-olivaceous (Flora of British Fungi Colour Identification Chart, 1969, Edinburgh: HMSO) mycelium with a white lobate margin at 24xc2x0 C. on 2% malt extract agar with glucose and peptone (MEA: composition per liter of distilled water: Difco malt extract, 20 g; Bacto-peptone, 1 g; agar, 20 g). After 7 days the mycelium attains a diameter of 2.5-3.5 cm.
Conidiomatal development is stimulated by exposure to near-UV light. Conidiomata are solitary, carbonaceous, unilocular, ostiolate and measure 1.5-2.0 mm wide and 1.25-2.0 mm high. Conidiogenous cells are borne on branched conidiophores which line the conidiogenous cavity. These cells are hyaline, obclavate to cylindrical, integrated, phialidic and measure 16-20 xcexcmxc3x971.5-2.0 xcexcm. Conidia are hyaline, aseptate and generally of three types: A-conidia (5.5-7.5 xcexcmxc3x971.5-3.0 xcexcm) are ellipsoid to fusiform, usually with acute apices and a guttule at each end; B-conidia (20-32 xcexcmxc3x97 less than 1 xcexcm) are hamate and filiform; C-conidia (9.5-11.5 xcexcmxc3x971.5-3.0 xcexcm) are obclavate with acute apices and usually at least three guttules. All three conidial types can be found within a single conidioma. The fluvial origin and observed microscopical characters did not allow further classification to species.
The 5,6-dihydro-xcex1-pyrones of formula (I) are associated primarily with the mycelium on termination of the fermentation. They may be recovered and purified from the medium. The separation and purification of the compounds from the fermentation broth and their recovery can be achieved using solvent extraction followed by chromatographic fractionation. The 5,6-dihydro-xcex1-pyrone of formula (I) in which R is CO2H may be converted into pharmaceutically or veterinarily acceptable salts by conventional methods. Suitable salts include salts with alkali metals such as sodium or potassium and ammonium salts.
The 5,6-dihydro-xcex1-pyrone of formula (I) wherein R is CH3 can, alternatively, be produced by the esterification of the phomalactone of formula (II): 
with (6S)-4,6-dimethyldodeca-2E,4E-dienoic acid which is a fatty acid of formula (IIIa): 
Preferably the reaction is carried out in the presence of a dehydrating agent such as DCC (dicyclohexylcarbodiimide) or EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and dimethylaminopyridine. The reaction is typically carried out in an inert solvent such as dichloromethane or tetrahydrofuran.
The reagents are generally mixed with stirring for example at a low temperature such as xe2x88x9278xc2x0 C. The reaction is then allowed to warm to room temperature (20-25xc2x0 C.) and stirred until complete. The reaction may be monitored chromatographically by thin layer chromatography or reversed phase high performance liquid chromatography and is typically complete within sixteen hours. Other dehydrating agents such as an alkylchloroformate and triethylamine, phenyldichlorophosphate, 2-chloro-1,3,5-trinitrobenzene and pyridine, and chlorosulphonyl isocyanate can also be used under similar conditions.
Alternatively an excess of the phomalactone of formula (II) can be reacted with the acid of formula (IIIa). The water formed can be removed by azeotropic distillation. Suitable solvents include toluene and 1,4-dioxane. The reaction is typically catalysed by acids such as sulphuric acid and ptoluenesulphonic acid.
It is advantageous to produce the 5,6-dihydro-xcex1-pyrone of formula (I) wherein R is CH3 by this route as both the phomalactone and the fatty acid can be produced by fermentation in larger quantities than the 5,6-dihydro-xcex1-pyrone of formula (I) wherein R is CH3.
The fatty acid of formula (IIIa) is one of a group of fatty acids of the following formula (III): 
wherein one of R1 and R2 is H and the other is H or OH. These fatty acids can be obtained by fermentation of the fungal strain Phomopsis sp 22502 (CBS 313.96) or a mutant thereof. In accordance with the present invention, therefore, the fatty acid of formula (III) can be produced by a process which comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, strain Phomopsis sp. 22502 (CBS 313.96) or a mutant thereof which produces the said fatty acid; and
(ii) isolating the said fatty acid of formula (III) from the fermentation broth.
The fatty acid of formula (III) may, of course, be isolated from the same fermentation broth as the 5,6-dihydro-xcex1-pyrones of formula (I). The fatty acid, like the 5,6-dihydro-xcex1-pyrones, is primarily associated with the mycelium on termination of fermentation.
Some of the fatty acids of formula (III) are novel. The present invention therefore further provides a fatty acid of formula (IIIb): 
wherein one of R1 and R2 is H and the other is OH
The phomalactone of formula (II) can be synthesised by methods known in the prior art, for example those disclosed in Krivobok, S. et al, Pharmazie, (1994): 49, H8, 605-607; Guirand, P. et al, Pharmazie, (1994): 49, H8, 279-281; Krasnoff, S. B. et al, J. Chem. Ecol., (1994): 20, 293-302 and Murayama, T. et al, Agric. Biol. Chem., (1987): 51, 2055-2060.
The present invention does however provide a new process for the preparation of the phomalactone of formula (II), which process comprises:
(i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain Paecilomyces sp. 3527 (CBS 314.96) or a mutant thereof which produces the said phomalactone; and
(ii) isolating the said phomalactone from the fermentation broth.
The phomalactone is found primarily in the culture liquor on termination of the fermentation and may be recovered and purified. The separation and purification of the compound from the fermentation broth and its recovery can be achieved using solvent extraction followed by application of conventional chromatographic fractionations with various chromatographic techniques and solvent systems.
The phomalactone of formula (II) has been isolated from a microorganism which we have designated X3527 and which has been identified as a strain of the genus Paecilomyces Bainier on the basis of the following morphological data with reference to the descriptions given by SAMSON, R. A., 1974 (Paecilomyces and some allied Hyphomycetes. Studies in Mycology No. 6. Baarn: CBS). The fungal strain Paecilomyes sp. (X3527) (CBS 314.96) is an entomogenous hyphomycete isolated from a tropical Lepidoptera pupa which produces white mycelium attaining 4.5-5.0 cm diameter within 14 days at 25xc2x0 C. on 2% MEA. The aerial mycelium becomes powdery as conidiogenesis occurs and may develop denser concentric zones. A pale-buff-yellow pigmentation frequently develops in aerial and/or submerged mycelium.
Conidiophores are hyaline and smooth-walled with stipe dimensions of 100-400 xcexcmxc3x971.5-2.5 xcexcm and bear single or sparsely clustered phialides. Phialides are produced with the characteristic morphology of Paecilomyces Bainier measuring 7-20 xcexcm long with a lower section inflated to 2-2.5 xcexcm wide. The phialide neck is often considerably attenuated ( less than 1 xcexcm wide) and bent. Phialides of very variable morphology are also produced, e.g. lacking an inflated basal region and/or with an attenuated neck measuring about 20 xcexcm long. Conidia are ellipsoid to cylindrical (3.5-7 xcexcmxc3x972-3 xcexcm), smooth-walled, hyaline and borne in conspicuous imbricate dry chains.
Further classification as a Paecilomyces anamorph of Cordyceps (Fries) Link may be justified on the grounds of the epidopterous origin of the isolate coupled with the results of numerous studies made by H. C. Evans and R. A. Samson (unpublished data) of entomopathogenic Paecilomyces anamorphs of Cordyceps.
The strains X22502 and X3527 were deposited by Xenova Group plc of 240 Bath Road, Slough, Berkshire, SL1 4EF, United Kingdom under the Budapest Treaty at the Centraalbureau voor Schimmelcultures, Baarn, the Netherlands, on 19th Mar. 1996 under references X07/64/502 and X08/64/527 respectively. Strain X22502 was assigned the reference number CBS 313.96. Strain X3527 was assigned the reference number CBS 314.96.
The present invention also embraces mutants of the above microorganisms. For example, those which are obtained by natural selection or those produced by mutating agents including ionising radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments, are also included within the ambit of this invention.
The invention further provides a biologically pure culture of fungal strain X22502 or X3527 or of a mutant thereof which produces the compounds of the invention. Such cultures are substantially free from other microorganisms. The invention also provides a process for fermenting the fungal strain X22502, X3527 or a said mutant, which process comprises fermenting strain X22502 or X3527 or a said mutant thereof in a source of carbon, nitrogen and inorganic salts.
Assimilable sources of carbon, nitrogen and minerals may be provided by either simple or complex nutrients. Sources of carbon will generally include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium.
Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers"" solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used. Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium.
Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.
An antifoam may be present to control excessive foaming and added at intervals as required.
Fermentation can be conducted at temperatures ranging from 20xc2x0 C. to 40xc2x0 C., preferably 24-30xc2x0 C. For optimal results, it is most convenient to conduct these fermentations at a temperature in the range 24-26xc2x0 C. The starting pH of the nutrient medium suitable for producing the compounds can vary from 5.0 to 8.5 with a preferred range of from 5.0 to 7.5.
Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask by known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of the fungal strain, loosely stoppering the flask with cotton wool, and permitting the fermentation to proceed in a constant room temperature of about 25xc2x0 C. on a rotary shaker at from 95 to 300 rpm for 2 to 10 days. The fermentation may also be conducted in static culture on liquid or semi-solid medium.
For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. The nutrient medium is made up in the tank after sterilization and is inoculated with a source of vegetative cellular growth of the fungal strain. The fermentation is allowed to continue for from 1 to 8 days while agitating and/or aerating the nutrient medium at a temperature in the range 20xc2x0 C. to 37xc2x0 C. The degree of aeration is dependent upon several factors such as the size of the fermenter and agitation speed. Generally the larger scale fermentations are agitated at about 95 to 750 rpm and aerations of about 0.5 to 1.5 VVM (volumes of air per volume of medium per minute).
The separation of the present compounds from the whole fermentation broth and their recovery is carried out by solvent extraction followed by application of chromatographic fractionations with various chromatographic techniques and solvent systems. The present compounds in pure form have thus been isolated in this way.
The 5,6-dihydro-xcex1-pyrones of formula (I) and pharmaceutically and veterinarily acceptable salts of the compound of formula (I) wherein R is CO2H are inhibitors of the production of cytokines, specifically IL-1xcex2.
These compounds can therefore be used in the treatment of disorders requiring immunosuppression, for example immunoinflammatory conditions and CNS disorders. A human or animal, e.g. a mammal, can therefore be treated by a method comprising administration of a therapeutically effective mount of a compound of formula (I), or a pharmaceutically or veterinarily acceptable salt of the compound of formula (I) wherein R is CO2H.
These compounds can be used in the treatment of an immunoinflammatory condition such as rheumatoid arthritis, osteoarthritis, septic shock, psoriasis, atherosclerosis, inflammatory bowel disease, Crohn""s disease and asthma. The compounds of the present invention also exhibit pharmacological properties associated with the treatment of other disorders requiring immunosuppression, for example central nervous system (CNS) disorders such as encephalomyelitis and Alzheimer""s disease.
The compounds of the present invention can be administered in a variety of dosage forms, for example orally such as in the form of tablets, capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example intramuscularly, intravenously or subcutaneously. The present compounds may therefore be given by injection or infusion.
The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Typically, however, the dosage adopted for each route of administration for adult humans is 0.001 to 10 mg/kg, most commonly in the range of 0.01 to 5 mg/kg, body weight. Such a dosage may be given, for example, from 1 to 5 times daily orally or by bolus infusion, infusion over several hours and/or repeated administration.
The toxicity of the compounds of the invention is negligible, they can therefore safely be used in therapy.
The compounds of the present invention are formulated for use as a pharmaceutical or veterinary composition also comprising a pharmaceutically or veterinarily acceptable carrier or diluent. The compositions are typically prepared following conventional methods and are administered in a pharmaceutically or veterinarily suitable form.
For example, the solid oral forms may contain, together with the active compound, diluents, such as lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dye-stuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates. Such preparations may be manufactured in known manner, for example by means of mixing, granulating, tabletting, sugar coating, or film coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose. The suspensions and the emulsion may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol.
Suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride. Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water which is generally Water for Injection. Preferably, however, they may take the form of a sterile, aqueous, isotonic saline solution. Alternatively, the compounds of the present invention may be encapsulated within liposomes.